Technical Abstract:
The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. These techniques involve the production of full-length cDNA libraries as a source of plasmid-based clones to express the desired protein in active form for determination of its function. These clones can be used in mutagenesis strategies to modify proteins for optimized functions. Plasmid-based functional proteomics requires rapid plasmid preparation methods to obtain adequate quantities of high-quality plasmid DNA to conduct all required steps in the process from creation of plasmid libraries to functional testing of expressed proteins. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. The ideal system would be an automated integrated programmable workcell capable of producing full-length cDNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. Such an automated system requires the integration of different equipment and instruments with the desired capabilities.
An example of this system is the integrated plasmid-based proteomic workcell built at USDA, NCAUR in Peoria, Illinois. The workcell automates all the required tasks from plasmid library creation through functional testing of the expressed protein(s) for large sets of clones. The robotic workcell was designed and built by USDA and Hudson Control Group, Inc. to conduct plasmid-based functional proteomics for optimization of gene ORF encoding proteins of interest for improved bioenergy applications.